Implications of increased S100β and Tau5 proteins in dystrophic nerves of two mdx mouse models for Duchenne muscular dystrophy

Dystrophic Dmd rats show early neuronal changes (increased S100β and Tau5) at 8 months, supporting severe dystropathology in this rodent model of Duchenne muscular dystrophy

“…These observations have been widely confirmed by others in mdx mice and also dystrophic dogs (Haddix et al, 2018;Marques et al, 2005; J o u r n a l P r e -p r o o f Journal Pre-proof 2007; Torres and Duchen, 1987). It is logical to consider that these early NMJ changes might subsequently impact the nerves that innervate these dystrophic muscles and this was confirmed by our recent study of dystrophic nerves from mdx mice aged from 13M up to 18M (Krishnan et al, 2020). These changes in dystrophic nerves suggest irreversible neurodegeneration and represent a secondary longer-term event in the disease progression with likely impaired neuronal function over time, that may become pronounced over many years of ongoing dystropathology in DMD boys and contribute further to the loss of dystrophic muscle function.…”

“…Many studies have focused on the dystrophic skeletal muscles, but recently we described changes in dystrophic nerves of mdx mice, with S100β and Tau5 neuronal proteins significantly increased in dystrophic sciatic nerves by 13 months (M) of age (Krishnan et al, 2020). This is far earlier than in normal wildtype (WT) mice where age-related neurodegenerative changes starts to manifest from about 15M-18M of age (Krishnan et al, 2016).…”

“…The mdx mouse is a widely used laboratory animal model for DMD, although the disease is mild compared with DMD, likely due to large phenotype differences between the species; this includes the relatively very short growth phase of mice (intensive for 6 weeks postnatal and over by ~3M) compared with humans (about 18 years), the short life span (~2 years for normal mice) and small size of mice (~20-30 gm weight) with far less muscle loading for this quadruped (Grounds, 2008;Willmann et al, 2009) .Many variations of mdx mouse models have been explored to try and produce a more severe phenotype that more closely resembles DMD [(reviewed in (van Putten et al, 2020)]. When we compared classic mdx mice with D2.mdx mice (on a DBA.2 background strain) that were proposed to be a more severe disease model, the surprising lack of change in D2.mdx nerves at 13M in contrast with classic mdx nerves, did not support severe ongoing myonecrosis and dystropathology in the D2.mdx mice (Krishnan et al, 2020). A more severe dystropathology occurs in various strains of dystrophic dogs including the classic Golden retriever muscular dystrophy (GRMD) model that more closely resemble the DMD boys (Kornegay, 2017).…”

“…These mice were aged at the Animal Resources Centre in Western Australia and all experiments were conducted in accordance with the National Health and Medical Research Council (Australia) guidelines approved by the Animal Ethics Committee of the University of Western Australia. Mice were killed by pentobarbital injection followed by cervical dislocation, sciatic and radial nerves dissected carefully away from the surrounding muscles and connective tissues and snap frozen in liquid nitrogen for future protein extraction (Krishnan et al, 2020). Due to the J o u r n a l P r e -p r o o f Journal Pre-proof smaller size of mouse nerves compared with rat nerves, both left and right mouse nerves were pooled for protein extraction (and thus tissue was not available for RNA analyses).…”

“…Protein extraction from the nerves were carried out as described in our previous studies (Krishnan et al, 2020;Krishnan et al, 2016;Krishnan et al, 2017). In short the snap frozen nerves were homogenized in buffer containing 20mM HEPES, 4% (w/v) sodium dodecyl sulphate (SDS) supplemented with protease and phosphatase inhibitor tablets followed by sonication for 10 seconds and centrifugation.…”